Device for extracting plasmid DNA from bacteria

Abstract

The application discloses a device for extracting plasmid DNA in bacteria and relates to the technical field of biological medicine. The device comprises a first mixing assembly and a second mixing assembly; the first mixing assembly is connected with the second mixing assembly through a lysis spiral pipe; at least one liquid inlet is arranged on a connecting pipeline of the lysis spiral pipe and the second mixing assembly; the device has the advantages that the mixing assembly is connected in series, the lysis and neutralization processes are carried out in a closed environment, the probability of polluting the environment is reduced, the device is convenient to clean after use, continuous processing is realized, and the production efficiency is improved; and the device is simple, and the design and production cost are small.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an apparatus for extracting plasmid DNA from bacteria. Background Technology

[0002] In recent years, due to the clinical success of gene therapy and DNA vaccines, the demand for industrial-scale plasmid fermentation production has become very urgent.

[0003] Current large-scale plasmid production technologies mainly involve the following steps: vector construction, bacterial fermentation, cell lysis, solid-liquid separation and clarification, and plasmid purification. While current plasmid production processes can produce plasmids that meet pharmaceutical quality standards and clinical requirements, several insurmountable bottlenecks remain. These include difficulties in achieving large-scale production (kilogram level), issues with vector copy number and stability, DNA denaturation during lysis, removal of residual HCD, challenges in solid-liquid separation, and endotoxin residue.

[0004] Plasmid DNA used in biopharmaceuticals is mainly produced in *E. coli*, and alkaline lysis is one of the most widely used methods for preparing plasmid DNA. This method utilizes alkaline conditions to lyse cells, causing irreversible denaturation of chromosomal DNA, while plasmid DNA is renatureable when the pH returns to neutral, thus separating the plasmid from the chromosomal DNA. The first and most crucial step in plasmid renaturation is cell lysis. The core issue in cell lysis is ensuring complete cell lysis, complete co-precipitation of chromosomal DNA, and removal of most of the RNA. Currently, the main problems with industrial-scale plasmid extraction processes include: 1. Lack of automated alkaline lysis equipment or equipment inadequate for thorough mixing; 2. Use of large amounts of organic solvents and acids, increasing safety risks and requiring sophisticated plant and equipment in large-scale industrial production; 3. Lack of automated mixing equipment or equipment inadequate for achieving uniform and low-shear mixing after mixing with neutralization solution (Solution III); 4. High solid-liquid separation costs in the neutralization reaction; 5. High levels of residual host DNA; 6. Low RNA removal rates, affecting downstream purification. 7. Reusable piping and equipment are difficult to clean, which is not conducive to CIP cleaning, while disposable consumables and equipment are expensive.

[0005] Chinese patent CN205710712U discloses a lysis device for plasmid extraction, including a frame, at least one receiving chamber rotatably mounted on the frame, the receiving chamber having a cavity for holding at least one bottle containing culture medium, a drive component for driving the receiving chamber to rotate, and a controller for controlling the drive component to drive the receiving chamber to rotate the bottle at a desired speed, direction, and number of revolutions. This technical solution is a relatively traditional processing method, suitable for laboratory production. Although the structure is relatively simple, the production speed is slow and continuous operation is not possible.

[0006] Chinese patent CN111733060A discloses a plasmid DNA alkaline lysis device, including a bacterial culture pipeline; a buffer solution pipeline; a bacterial culture suspension circulation device, including a circulation pipeline connected end-to-end to form a loop, connecting the bacterial culture pipeline and the buffer solution pipeline; a pump and valves are installed on the circulation pipeline; an alkaline lysis buffer pre-mixing pipeline; a cell lysis reactor; a neutralization reactor; an acid solution pipeline; and a collection device. This technical device achieves continuous lysis, and with appropriate filtration devices, it can effectively improve production levels. However, this technical solution still requires aeration for foaming and mixing, has a complex structure, and features interwoven pipelines, which is not conducive to pipeline cleaning.

[0007] Chinese patent CN111808716A discloses a plasmid extraction device, including a lysis container, a precipitation container, an eluent container, a collection container, and a chromatography column. The lysis container and the precipitation container are connected by a first connecting pipe, the precipitation container is connected to the chromatography column by a second connecting pipe, and the eluent container is connected to the chromatography column by a third connecting pipe. Valves are installed on the first, second, and third connecting pipes. The collection container is located below the chromatography column, and the chromatography column is connected to a vibration mechanism. The above technical solution adopts a vibration-based structure, resulting in a discontinuous processing process that requires further improvement in processing efficiency. Furthermore, this method suffers from a high level of host DNA residue, necessitating further improvement.

[0008] Although we previously developed a method for extracting plasmid DNA from bacteria by lysing and neutralizing it using a mixing chamber oscillation (see: 200610114061.6, A method for continuous large-scale plasmid extraction), this method is not easy to scale up and has low plasmid DNA extraction efficiency. To solve this problem, we developed a device for extracting plasmid DNA using a bubble mixer (see: 202011120617.9, A bubble generating device for extracting plasmid DNA from bacteria). This device can homogenize and fully mix the bacterial solution and the lysis buffer. The bubble mixing effectively reduces shear force and improves yield and quality. However, this device still has the problem of not being easy to scale up. It is necessary to customize bubble mixers of different sizes according to the scale and explore the scale-up conditions such as aeration rate and flow rate.

[0009] Therefore, we have invented and designed a novel plasmid DNA extraction device and method. Besides effectively controlling the shear force during the mixing process, it facilitates scale-up of the process. Compared to methods using bubble mixers (bubble generators), it effectively improves the mixing efficiency of the lysis and neutralization processes, increasing the yield. Furthermore, by easily adjusting the mixing parameters, it effectively controls the shear force, improving plasmid quality. The device is also simple in principle and allows for precise control, thus shortening the time required to establish scale-up conditions and further improving work efficiency. This will greatly promote the development of research related to continuous, large-scale plasmid DNA extraction, and is of significant importance. Summary of the Invention

[0010] In view of this, the present invention provides an apparatus for extracting plasmid DNA from bacteria, which can solve the above problems.

[0011] To this end, the present invention is implemented by the following technical solution.

[0012] On one hand, the present invention provides an apparatus for extracting plasmid DNA from bacteria, comprising: a first mixing component and a second mixing component;

[0013] The first mixing component and the second mixing component are connected through the pyrolysis spiral tube; at least one liquid inlet is provided on the connecting pipe between the pyrolysis spiral tube and the second mixing component;

[0014] After the resuspended bacterial solution flows into the first mixing component and is mixed, it is pyrolyzed through the pyrolysis spiral tube to obtain a lysate, which is then introduced into the second mixing component to neutralize with solution III to obtain a neutralization reaction solution. The lysate enters the second mixing component through the inlet.

[0015] Furthermore, the structures of both the first mixing component and the second mixing component can be one of the following: stirring type, emulsifying type, and centrifugal type.

[0016] Furthermore, the first mixing component has a stirring or emulsifying structure; the second mixing component has a centrifugal structure. Preferably, both the first and second mixing components are mixing pumps or agitators; more preferably, the first and second mixing components are respectively a first mixing pump and a second mixing pump, and the impellers of both the first and second mixing pumps are preferably semi-closed impellers. By adopting the form of a mixing pump, the pyrolysis and neutralization processes are carried out in a closed environment, reducing the probability of environmental pollution, and facilitating CIP and SIP after use.

[0017] Furthermore, both the impellers of the first mixing pump and the second mixing pump can include a rear cover plate; a plurality of guide columns are evenly distributed on the rear cover plate, and the outer surface of the guide columns is arc-shaped at least along the rotation direction of the impeller; the guide columns are preferably one or more combinations of cylindrical, frustum-shaped or fan-shaped columns.

[0018] Furthermore, the diameter of the flow-guiding column is 0.5mm-40mm, preferably 2mm-10mm; the flow-guiding column is preferably cylindrical, or the cross-sectional area of ​​the flow-guiding column is largest in the middle and gradually decreases from the middle to both ends. Multiple flow-guiding columns are evenly distributed, and their diameters are within a suitable range, which can reduce shear force, prevent host DNA contamination of the product, and enable automated lysis and neutralization.

[0019] Furthermore, the cross-sectional area of ​​the guide column is the largest in the middle, and gradually decreases from the middle to both ends. In specific implementation, the structure of the guide column can be spindle-shaped.

[0020] Furthermore, the inner diameter of the pyrolysis spiral tube is 0.5cm-15cm, preferably 0.5cm-9cm; the pump head diameter of the first mixing pump and the second mixing pump can both be 2cm-100cm, preferably 4cm-30cm; the ratio of the pump chamber volume of the first mixing pump and the second mixing pump to the rated feed volume per minute of a single mixing pump can both be in the range of 1:6-1:1, preferably 1:6-1:3; or the pump chamber volume of the first mixing pump and the second mixing pump is the volume of the liquid flowing through the pump chamber for 10s-60s, preferably the volume of the liquid flowing through the pump chamber for 10s-20s.

[0021] Furthermore, the length of the guide columns is related to their distribution position, with the length of each guide column decreasing sequentially from the center of the rear cover plate to the outer edge, and the apex of each guide column located on the same parabolic surface.

[0022] Furthermore, the inlet and outlet ends of both the first and second mixing pumps can be coaxially arranged; the inlet end is located at the center of the pump casing, and the outlet end is located at the center of the pump base. This ensures that the fluid entering the pump chamber must pass through the rear cover plate from the center to the edge, winding around to the rear before being discharged, allowing it to fully contact the guide column and achieve uniform mixing, thus improving the quality of the neutralization reaction.

[0023] Furthermore, the device also includes a filtration assembly, with the outlet of the second mixing assembly connected to the inlet of the filtration assembly, through which the neutralization reaction solution is filtered.

[0024] Furthermore, the resuspended bacterial solution includes solution I and bacterial cells containing plasmid DNA. The resuspended bacterial solution is mixed and transported to the first mixing component by a first delivery pump, and then mixed with solution II which is transported to the first mixing component by a second delivery pump before being passed into a lysis spiral tube for lysis.

[0025] Furthermore, the filter assembly structure is one or more combinations of screen type, depth filtration type, and centrifugal filtration type.

[0026] Furthermore, the filter assembly has a screen-type or deep-layer filtration structure; the filter pore size is 0.2μm-800μm, specifically 0.2μm-200μm; the filter material includes, but is not limited to, cellulose, diatomaceous earth, activated carbon, polypropylene fiber, and silica gel.

[0027] Furthermore, the filter assembly has a centrifugal structure; the centrifugal force is 1000g-20000g, the centrifugation time is 2min-60min, and the temperature is 2℃-40℃.

[0028] On the other hand, the present invention also provides a method for extracting plasmid DNA from bacteria using the above-described apparatus, wherein lysis and neutralization during plasmid production are achieved in two tandem hybrid components, specifically including the following steps:

[0029] Step S1: Mix;

[0030] Step S2: Pyrolysis;

[0031] Step S3: Neutralize;

[0032] Step S1 is completed in the first mixing component, step S2 is completed in the pyrolysis spiral tube, and step S3 is completed in the second mixing component. The first mixing component, the pyrolysis spiral tube, and the second mixing component are connected in series.

[0033] Furthermore, in step S2, the pyrolysis lasts for 2-10 minutes, preferably 5 minutes.

[0034] Furthermore, the rotational speed of the first mixing component is 50 rpm to 1500 rpm, preferably 200 rpm to 500 rpm; the rotational speed of the second mixing component is 20 rpm to 1000 rpm, preferably 150 rpm to 500 rpm.

[0035] Furthermore, the method for extracting plasmid DNA from bacteria specifically includes the following steps:

[0036] Step S1: After resuspending the bacterial cells with solution I, a resuspended bacterial solution is obtained. Then, the resuspended bacterial solution and solution II are introduced into the first mixing component for mixing to obtain a bacterial cell mixture.

[0037] Step S2: The bacterial mixture flows out from the first mixing component and enters the lysis spiral tube for lysis, and the lysate is obtained after lysis;

[0038] Step S3: After the pyrolysis solution and solution III enter the second mixing component, a neutralization reaction is carried out (or the pyrolysis solution and solution III are premixed before being introduced into the second mixing component) to obtain a neutralized reaction solution;

[0039] Preferably, after obtaining the neutralization reaction solution, the process further includes a step of solid-liquid separation and purification.

[0040] in,

[0041] In step S1, the volume-to-mass ratio of the resuspended bacterial solution to the bacterial cells is 3-20:1 (L:kg), more preferably 7:1 (L:kg).

[0042] In step S1, the volume ratio of solution I to solution II is 1:0.5-1:3, more preferably 1:1. By varying the diameter and length of the tubing, the alkaline lysis time is controlled to be 2-10 minutes to ensure complete cell lysis and optimal lysis effect.

[0043] In step S1, solution I comprises Tris-HCl and EDTA-2Na. More preferably, the concentration of Tris-HCl is 2 mmol / L-100 mmol / L, the concentration of EDTA-2Na is 0.1 mmol / L-50 mmol / L, and the pH range of solution I is 6.0-9.0.

[0044] In step S1, solution II comprises NaOH and SDS. More preferably, the concentration of NaOH is 0.02 mol / L-5 mol / L, and the concentration of SDS is 0.1%-10%.

[0045] In step S2, the pyrolysis time is 2 min to 10 min, more preferably 5 min.

[0046] In step S3, solution III includes KAc and NH4Ac. More preferably, the concentration of KAc is 0.1 mol / L-6 mol / L and the concentration of NH4Ac is 0.2 mol / L-10 mol / L.

[0047] In step S3, the volume ratio of lysis buffer to solution III is 1:0.3-5, more preferably 1:1. These conditions are used to control the lysis and neutralization effects, ensuring the precipitation of host DNA and the removal of host RNA.

[0048] The present invention has the following advantages:

[0049] This invention innovatively adopts a series of mixing components in the alkaline pyrolysis and neutralization stages of plasmid production, combined with a delivery pump, to ensure that the pyrolysis and neutralization processes are carried out in a closed environment, reducing the chance of environmental pollution. After use, it facilitates CIP and SIP, and enables continuous processing, improving production efficiency. It also facilitates the scaling up of production. Compared with the current mainstream airmix bubble mixer production system, it is easier to scale up, does not require the customization of bubble mixers of different sizes according to the scale, shortens the time for exploring scale-up conditions, and improves work efficiency.

[0050] Furthermore, the equipment of this invention is simple, easy to operate, and inexpensive. It does not require specialized, customized, or expensive equipment, making it easy to scale up in production and reducing production costs. The two mixing components used ensure thorough mixing of the bacterial culture and lysis buffer while also ensuring gentle mixing and neutralization of the neutralizing solution, avoiding the use of complex low-shear neutralization equipment. During lysis, mixing is thorough and the mixing time is short, while the conditions during neutralization are mild and uniform. After lysis and neutralization, the residual host DNA and RNA are lower than those achieved by a bubble mixer, resulting in high product quality. At the same time, the size of the pump chamber is optimized, making the lysis and neutralization time and shear force suitable for product production. The proportion of supercoiled plasmids after lysis is high, and the residual host DNA and RNA are low. In addition, there is no need to use a complex multi-stage membrane filtration system, and there is no need for overnight precipitation after lysis. The equipment can be directly cleaned with CIP, which meets the production specifications for pharmaceutical manufacturing, while saving process time and reducing costs.

[0051] Furthermore, by optimizing the size of the pump chamber and adjusting the ratio of the pump chamber to the flow rate, the time and shear force for lysis and neutralization are made suitable for product production, while also facilitating the scaling up of production. The dimensions of the mixing pump head are optimized, and 3D printing technology is used to design and customize the pump head. While ensuring the mixing effect, the shear force is reduced, preventing host DNA from contaminating the product, and enabling lysis and neutralization to be automated.

[0052] Furthermore, the preparation process does not add high-risk animal-derived ingredients such as RNase, lysozyme, and proteinase K. The production process does not use toxic organic solvents such as isopropanol, phenol, anhydrous ethanol, and other mutagens. The reagents used can be general reagents or meet pharmaceutical grade requirements. No acid neutralization is used, and the requirements for factory equipment are low, making it suitable for large-scale production. Attached Figure Description

[0053] To more clearly illustrate the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. Obviously, the drawings described below are only one or several embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0054] Figure 1 This is a schematic diagram of the apparatus for extracting plasmid DNA from bacteria according to the present invention;

[0055] Figure 2 This is a perspective view of the impeller of the first hybrid component in Embodiment 1 of the present invention;

[0056] Figure 3 This is a perspective view of the second hybrid component in Embodiment 1 of the present invention;

[0057] Figure 4 for Figure 3 Exploded view of the second hybrid component in the diagram;

[0058] Figure 5 for Figure 3 Structural diagram of the second mixing component after removing the pump casing;

[0059] Figure 6 for Figure 3 A three-dimensional view of the impeller of the second mixing component;

[0060] Figure 7 This is a comparison diagram of the electrophoresis results of the product in Example 1 of the present invention, wherein lane 1: marker, lane 2: centrifugation supernatant, and lane 3: control;

[0061] Figure 8 This is a side view of the impeller of the second mixing component in Embodiment 2 of the present invention;

[0062] Figure 9 This is a perspective view of the second hybrid component in Embodiment 3 of the present invention;

[0063] Figure 10 This is a perspective view of the second hybrid component in Embodiment 3 of the present invention;

[0064] Figure 11 The above is a comparison diagram of the electrophoresis results of Examples 1, 4, and 5 of the present invention, wherein lane 1: supernatant of neutralization reaction solution of Example 5, lane 2: supernatant of neutralization reaction solution of Example 1, lane 3: supernatant of neutralization reaction solution of Example 4, lane 4: standard, and lane 5: marker.

[0065] Figure 12 The image shows the electrophoresis results of Comparative Example 1 of the present invention, wherein lane 1: supernatant of neutralization reaction solution of Example 1, lane 2: supernatant of neutralization reaction solution of Comparative Example 1, lane 3: standard, and lane 4: marker.

[0066] Figure 13 This is a perspective view of the impeller of the second mixing component in Comparative Example 2 of the present invention;

[0067] Figure 14 This is a perspective view of the impeller of the second mixing component in Comparative Example 3 of the present invention;

[0068] Figure 15 This is a comparison diagram of the electrophoresis results of Example 1 and Comparative Example 3 of the present invention, wherein lane 1: supernatant of neutralization reaction solution of Example 1, lane 2: supernatant of neutralization reaction solution of Comparative Example 3, lane 3: standard, lane 4: marker;

[0069] Figure 16 This is a perspective view of the flow guide column in Embodiment 6 of the present invention;

[0070] in, Figure 1-6 , 9-10 and 16:

[0071] 1-First mixing component; 2-Second mixing component; 202-Pump base; 203-Sealing ring; 204-Impeller; 205-Pump casing; 2021-Annular groove; 2041-Rear cover plate; 2042-Guide column; 3-Cycling spiral tube; 4-Filter assembly; 5-Resuspension of bacterial solution; 6-Solution II; 7-Solution III; 201-Main shaft. Detailed Implementation

[0072] The following will provide a clear and complete description of the concept, specific structure, and technical effects of the present invention in conjunction with embodiments and accompanying drawings, so as to fully understand the purpose, solution, and effects of the present invention. It should be noted that, unless otherwise specified, the features in the embodiments of this application can be combined with each other.

[0073] It should also be noted that, in the description of this invention, unless otherwise explicitly specified and limited, the terms "set," "install," "connect," "link," etc., should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0074] Unless otherwise specified, "lysis solution" in this article refers to "solution II".

[0075] Unless otherwise specified, "neutralizing solution" in this article refers to "solution III".

[0076] The invention will now be further described with reference to the accompanying drawings.

[0077] like Figure 1 As shown, the apparatus for extracting plasmid DNA from bacteria according to the present invention includes: a first mixing component 1, a second mixing component 2, and a filtering component 4. The two mixing components are distinguished by function: the first mixing component 1 can be a lysis mixing component, and the second mixing component 2 can be a neutralization mixing component.

[0078] The first mixing component 1 and the second mixing component 2 are connected in series; a lysis helical tube 3 is also connected in series between the two mixing components. Specifically, the solution I required for the lysis reaction is mixed with bacterial cells containing plasmid DNA to form a resuspended bacterial solution 5, which is then delivered to the first mixing component 1 (i.e., the lysis mixing pump) by controlling the flow rate and velocity through the first delivery pump. In specific implementation, a first tee connector (i.e., a "Y"-shaped connector) is also connected in series on the delivery pipeline. The resuspended bacterial solution 5 and solution II 6 can be delivered to the first tee connector through the first delivery pump and the second delivery pump, respectively, and then introduced into the first mixing component 1 for mixing to obtain a bacterial cell mixture.

[0079] The outlet of the first mixing component 1 is connected to the inlet of the pyrolysis spiral tube 3; the outlet of the pyrolysis spiral tube 3 is connected to the inlet of the second mixing component 2. Specifically, an inlet is provided on the pipeline between the pyrolysis spiral tube 3 and the second mixing component 2, which is a second tee connector in the series pipeline, i.e., a "Y"-shaped connector. One end of the second tee connector is also connected to the container of solution Ⅲ7 via a third delivery pump.

[0080] Preferably, the first mixing component 1 and the second mixing component 2 used in this embodiment can be a stirrer or a mixing pump, specifically a first mixing pump and a second mixing pump, including but not limited to stirring pumps, emulsifying pumps, centrifugal pumps, etc. The stirring pump's impeller can be a paddle stirrer, propeller stirrer, turbine stirrer, anchor stirrer, frame stirrer, or spiral stirrer; the emulsifying pump's rotor and stator include, but are not limited to, coarse-tooth, medium-tooth, and fine-tooth. Through a pump head of a certain regular shape, thorough mixing of the solution is achieved, and the shear force is low, ensuring that the chromosomal DNA does not undergo extensive breakage, and the process can be carried out in a closed environment without contamination. Furthermore, since the first mixing component 1 is used for the lysis reaction, the structure of the first mixing component 1 is preferably one of stirring or emulsifying, specifically an emulsifying pump, whose blade structure can be as follows... Figure 2 The example shown (and may also take the form of other emulsifying pumps in the prior art, but only one is shown here) Figure 2 (As shown); the second mixing component 2 is used for the neutralization reaction and can be selected as a centrifugal structure, such as... Figure 3 , 4 As shown, the second mixing assembly 2 mainly includes a main shaft 201, a pump base 202, a sealing ring 203, an impeller 204, and a pump casing 205. The fluid transport route is as follows: Figure 3As indicated by the middle arrow, fluid enters the pump through the inlet end at the center of the pump casing 205. After centrifugal mixing, it flows out through the outlet end located laterally on the pump casing 205. The internal pipeline of the outlet end is tangential to the pump cavity. One end of the main shaft 201 is connected to the output end of an external motor, and the other end passes through the center of the pump base 202 via a sealing device and is fixedly connected to the impeller 204. An annular groove is machined in the contact area between the pump base 202 and the pump casing 205 for installing the sealing ring 203. The impeller 204 is preferably a semi-closed impeller; however, traditional impellers have significant drawbacks, namely, high shear force. Therefore, in this embodiment, the impeller 204 is... Figure 5 , 6 The design shown includes a rear cover plate 2041. A total of 32 guide columns 2042 are evenly distributed on the rear cover plate 2041, arranged in three layers around the center, and the guide columns 2042 are perpendicular to the surface of the rear cover plate 2041. Furthermore, to better reduce the generated shear force, the shape of the guide columns 2042 can be one or more combinations of cylinders, frustums, or fan-shaped structures, preferably cylinders. The diameter of the guide columns 2042 ranges from 0.5mm to 40mm; testing has shown that a diameter of 2mm to 10mm yields better results. Optimized design can reduce shear force, prevent host DNA contamination of the product, and automate lysis and neutralization. Experiments show that the second mixing component 2, by setting a certain rotation speed range, controls the mixing effect and shear force at different scales. Combined with the first mixing component 1, it can achieve automatic lysis and neutralization of bacterial solutions of different scales, thereby realizing continuous, large-scale production. In specific implementation, the structure of the first mixing component 1 can also be the same as that of the second mixing component 1. Specifically, the impeller speed of the second mixing component 2 produces a better mixing effect when it is between 20 rpm and 1000 rpm. The neutralization effect can also be controlled by changing the pump head shape, size, and speed of the second mixing component 2; the pump head diameter of the second mixing pump is 2 cm to 100 cm, preferably 4 cm to 30 cm, the speed is controlled between 20 rpm and 1000 rpm, preferably 150 rpm to 500 rpm, and the ratio of the pump chamber volume to the rated feed volume per minute of the mixing pump is between 1:6 and 1:1, preferably 1:6 to 1:3; or the pump chamber volume is designed to be the volume of the liquid flowing through the pump chamber for 10 s to 60 s, preferably 10 s to 20 s; ensuring complete neutralization and generating low shear force, reducing chromosomal DNA breakage, and improving the quality of plasmid DNA.

[0081] The outlet of the second mixing component 2 is connected to the inlet of the filter component 4.

[0082] Preferably, the filter assembly 4 has a structure that is one or a combination of screen type, depth filtration type, and centrifugal filtration type. Specifically, in this embodiment, the filter assembly 4 has a depth filtration structure; the filtration pore size is 0.2μm-800μm; specifically, the filtration pore size can be selected between 0.1μm-200μm. The supernatant after neutralization is clarified a second time through depth filtration. The filter material includes, but is not limited to, cellulose, diatomaceous earth, activated carbon, polypropylene fiber, silica gel, and combinations thereof. The coating area of ​​the depth filtration membrane is 0.01m². 2 -2m 2 between.

[0083] The work process is as follows:

[0084] Step S1: Solution I and bacterial cells containing plasmid DNA are premixed to obtain a resuspended bacterial solution. The resuspended bacterial solution 5 and solution II 6 can be sent to the first three-way connector by the first delivery pump for premixing in the pipeline. Then, they enter the first mixing component 1 for stirring and mixing to obtain a bacterial cell mixture.

[0085] Step S2: The stirred and mixed bacterial mixture enters the lysis spiral tube 3 for lysis reaction, and then flows out to obtain the lysate;

[0086] Step S3: The pyrolysis liquid flowing out of the pyrolysis spiral tube 3 is premixed with solution III 7 and then transported to the second mixing component 2 through the second three-way connector (or the pyrolysis liquid flowing out of the pyrolysis spiral tube 3 and solution III flowing through the second delivery pump are both transported to the second three-way connector and then introduced into the second mixing component 2) to undergo a neutralization reaction and obtain the neutralized reaction liquid.

[0087] Step S4: After neutralization, the neutralized reaction solution flows out and enters the filter assembly 4 for filtration, solid-liquid separation and purification.

[0088] in,

[0089] In step S1, the volume-to-mass ratio of the resuspended bacterial solution to the bacterial cells is 3-20:1 (v:m), more preferably 7:1 (v:m).

[0090] In step S1, the volume ratio of solution I to solution II is 1:0.5-1:3, and more preferably 1:1.

[0091] In step S1, solution I includes Tris-HCl and EDTA-2Na. More preferably, the concentration of Tris-HCl is 2 mmol / L-100 mmol / L, the concentration of EDTA-2Na is 0.1 mmol / L-50 mmol / L, and the pH range of solution I is 6.0-9.0.

[0092] In step S1, solution II comprises NaOH and SDS. More preferably, the concentration of NaOH is 0.02 mol / L-5 mol / L and the concentration of SDS is 0.1%-10%.

[0093] In step S2, the pyrolysis time is 2 min to 10 min, more preferably 5 min.

[0094] In step S3, solution III includes KAc and NH4Ac. More preferably, the concentration of KAc is 0.1 mol / L-6 mol / L and the concentration of NH4Ac is 0.2 mol / L-10 mol / L.

[0095] In step S3, the volume ratio of the lysis buffer flowing out of the lysis helical tube 3 to solution III is 1:0.3-5, more preferably 1:1. These conditions are used to control the lysis and neutralization effects, ensuring the precipitation of chromosomal DNA and the removal of host RNA.

[0096] In step S4, the solid-liquid separation method includes, but is not limited to, one or more combinations of filtration, depth filtration, and centrifugation. Preferably, when filtration is selected, the filtration material includes, but is not limited to, one or more of cellulose, diatomaceous earth, activated carbon, polypropylene fiber, and silica gel, with a pore size of 0.2 μm-800 μm. Preferably, when depth filtration is selected, the depth filtration material includes, but is not limited to, one or more of cellulose, diatomaceous earth, activated carbon, polypropylene fiber, and silica gel, with a pore size of 0.1 μm-200 μm and a membrane area of ​​0.01 m². 2 -2m 2 Preferably, when selecting centrifugation, the centrifugation method includes, but is not limited to, using a benchtop centrifuge, a tubular centrifuge, or a disc centrifuge, with a centrifugal force of 1000g-20000g, a centrifugation time of 2min-60min, and a centrifugation temperature of 2℃-40℃.

[0097] The extraction devices described in the above embodiments can all be used in the following embodiments, and differences will be shown in detail. A specific method for extracting plasmid DNA from bacteria is as follows:

[0098] Example 1: 50L Fermentation Scale Treatment Case

[0099] The apparatus for extracting plasmid DNA from bacteria in Example 1 has a pump head diameter of 10 cm, and the impellers of both pump heads are as shown in the attached diagram. Figure 6 As shown, the diameter of the guide column is 5mm.

[0100] (1) The OD of the high-density fermentation broth of Escherichia coli containing plasmid A was measured by spectrophotometer. 600The concentration was 84.2. 23.3 L of fermentation broth was centrifuged to harvest 3684 g of cells, with a wet weight of 15.8%. The 3684 g of cells were resuspended in a pH 8.0 resuspension solution (Solution I) consisting of 25 mM Tris-HCl and 10 mM EDTA-2Na to obtain a resuspended bacterial solution with a volume of 25.8 L (cell volume to resuspension volume weight-to-volume ratio of 1:7).

[0101] (2) The resuspended bacterial solution was pumped to the lysis mixing pump at a rate of 140 ml / min, while the lysis solution (solution II) consisting of 0.2 M NaOH and 1% SDS was pumped to the lysis mixing pump (first mixing component) at a rate of 140 ml / min. The speed of the lysis mixing pump was adjusted to 200 rpm, and lysis mixing was started to obtain the bacterial cell mixture. The pump chamber volume of the lysis mixing pump was 1:3 of the rated feed volume per minute of a single mixing pump.

[0102] (3) After the bacterial mixture is pumped out from the lysis mixing pump, it enters the lysis spiral tube 3. The inner diameter of the lysis spiral tube 3 is 1.9 cm and the length is 5 m. The lysis time in the lysis spiral tube 3 is 5 min to obtain the lysate.

[0103] (4) The pyrolysis liquid after pyrolysis enters the neutralization mixing pump (second mixing component). The other end of the neutralization mixing pump is supplied with solution III (pre-cooled at 2-8℃) composed of 1M KAc and 7M NH4Ac at a rate of 280 ml / min. The neutralization mixing pump is set to a speed of 250 rpm. The diameter of the guide column on the impeller of the neutralization mixing pump is 5 mm and the shape of the guide column is cylindrical. The diameter of the pump head of the neutralization mixing pump is 8.5 cm. The pump chamber volume of the neutralization mixing pump is 1:4 of the rated feed volume per minute of a single mixing pump.

[0104] (5) After neutralization, collect the neutralization reaction solution and enter the filter assembly 4. Select the centrifugal filtration method, centrifuge at 8000g for 20min and collect the supernatant for the next purification step.

[0105] Result detection:

[0106] The plasmid concentration in the resuspended bacterial culture was 545 mg / L (measured using a QIAGEN plasmid miniprep kit), and the total plasmid amount was 14.06 g.

[0107] After neutralization, 100 L of neutralization reaction solution was obtained. After centrifugation, 82 L of supernatant was obtained. The plasmid concentration was determined to be 121.8 mg / L by HPLC (HPLC model: Waters 2695; column model: TOSOH, Tskgel DNA-NPR 4.6 mm * 7.5 cm 2.5 μm; the HPLC determination conditions in the following examples were the same). The lysis yield was 71%.

[0108] See electrophoresis image Figure 7 ,from Figure 7 It can be seen that the plasmid purity in the supernatant after lysis using the apparatus and method of this application is high, while the RNA and host DNA content is low.

[0109] The plasmid DNA obtained by the above method was detected by HPLC and pharmacopoeia methods. The results showed that the plasmid was the target plasmid with high purity, a supercoil ratio of more than 95%, and a low open-circular ratio.

[0110] Example 2

[0111] Based on Example 1, in this example, the length and distribution position of the guide columns 2042 are related. The length of each guide column 2042 decreases sequentially from the center of the rear cover plate 2041 to the outer edge, and the apex of each guide column 2042 is located on the same parabolic surface. Figure 8 As shown by the dashed line. The inner surface of the corresponding pump casing 205 is also designed as a parabolic shape to match the guide column 2042.

[0112] The filter assembly 4 has a centrifugal structure and can be selected as a benchtop, tubular, or disc centrifuge connected in series; the centrifugal force is 1000g-20000g, the centrifugation time is 2min-60min, and the temperature is 2℃-40℃.

[0113] Example 3

[0114] Based on Example 1, in this example, the inlet and outlet ends of the second mixing component 2 are coaxially arranged, combined with... Figure 9 , 10 As shown, the inlet is located at the center of the pump housing 205, while the outlet is not located on the circumferential side of the pump housing 205. In order to further increase the mixing effect and avoid uneven mixing caused by the inlet and outlet being too close to each other, the outlet is located at the center of the pump base 202, that is, behind the rear cover plate 2041. An annular groove 2021 is machined around the rotating shaft, and an opening can be made at the bottom or side of the groove as the outlet. In this way, the fluid entering the pump cavity must pass through the rear cover plate 2041 from the center to the edge, and can only be discharged through the annular groove 2021 after going around to the rear, so that it can fully contact the guide column 2042, achieve the purpose of uniform mixing, and improve the quality of neutralization reaction.

[0115] Example 4

[0116] Unlike Example 1, the first mixing pump operates at 400 rpm, the second mixing pump operates at 500 rpm, and the diameter of the pyrolysis spiral tube is 1.9 cm; the pump head diameter of the second mixing pump is 10 cm. The guide column is cylindrical with a diameter of 1 mm. Everything else is the same.

[0117] Then, the neutralized reaction solution was subjected to agarose gel electrophoresis, and the test results were as follows: Figure 11 As shown, the host DNA and RNA in the lysate supernatant of Example 4 were higher than those in the lysate supernatant of Example 1, proving that there will be more impurities when the rotation speed is too high.

[0118] Example 5

[0119] Unlike Example 1, the bacterial resuspension was 2.5 L, the first mixing pump rotated at 100 rpm, the second mixing pump rotated at 50 rpm, and the diameter of the lysis spiral tube was 1.9 cm; the pump head diameter of the second mixing pump was 10 cm. The guide column was cylindrical with a diameter of 1 mm. Everything else was the same.

[0120] Then, 10 L of neutralization reaction solution was obtained after neutralization. Centrifugation yielded 7.8 L of supernatant. The plasmid concentration in the supernatant was measured to be 96 mg / L (HPLC determination), with a lysis yield of 55.0%. Electrophoresis results are as follows: Figure 11 As shown, from Figure 11 It can be seen that when the speed of the first and second mixing pumps is low, the mixing and neutralization will be insufficient, and the plasmid DNA yield will be lower than that in Example 1.

[0121] Example 6

[0122] Unlike Example 1, the guide column 2042 in this example features a variable cross-section design to further reduce the impact of shearing on the neutralization process. This is achieved through fluid motion analysis, such as... Figure 16 As shown by the middle arrow, the velocity distribution of the fluid relative to the main body during the rotation of a single guide column; that is, it decreases from the middle layer to both sides. The reason is that the fluid is subjected to viscous resistance from the pump casing (i.e., the pump base) on both the upper and lower sides, resulting in a gradient velocity distribution. Therefore, in order to maintain a relatively consistent shear force generated by the single guide column on the genetic material in the fluid, it is designed as a variable cross-section structure. Specifically, taking the cylinder in Example 1 as an example, the cross-section of the single guide column increases and then decreases from the pump casing side to the pump base side, forming a "spindle-shaped" structure. See details... Figure 16 Although the relative velocity and impact are relatively high at the center of the guide column 2042, the above design, combined with the large radius of curvature and the force-bearing area, can effectively reduce the shearing effect on the plasmid and improve the plasmid yield to a certain extent.

[0123] Comparative Example 1

[0124] Unlike Example 1, the neutralization step in Comparative Example 1 was carried out in a bubble mixer without the use of a pump, and the specific steps are as follows:

[0125] (1) The OD600 of the high-density fermentation broth of *E. coli* containing plasmid A was measured to be 78.9 using a spectrophotometer. 23.5 L of the fermentation broth was centrifuged, and 3603 g of cells were harvested, with a wet weight of 15.3%. The 3603 g of cells were resuspended in a pH 8.0 resuspension solution (solution I) composed of 25 mM Tris-HCl and 10 mM EDTA-2Na to obtain a resuspension broth with a volume of 25.2 L (the mass-to-volume ratio of cells to solution I was 1:7).

[0126] (2) The resuspended bacterial solution was pumped to one side of the "Y" connector at a rate of 140 ml / min, while the lysis solution (solution II) consisting of 0.2 M NaOH and 1% SDS was pumped to the other side of the "Y" connector at a rate of 140 ml / min. The "Y" connector was then connected to the lysis mixing pump (first mixing component), and the rotation speed was adjusted to 200 rpm to begin lysis mixing, resulting in a bacterial cell mixture. The volume ratio of solution I to solution II was 1:1.

[0127] (3) After the bacterial mixture is pumped out from the lysis mixing pump, it enters the lysis spiral tube. The inner diameter of the lysis spiral tube is 1.9 cm and the length is 5 m. The lysis time in the lysis spiral tube is 5 min to obtain the lysate.

[0128] (4) The pyrolysis solution enters another "Y"-shaped connector. The other end of the connector is supplied with solution III (pre-cooled at 2-8°C) consisting of 1M KAc and 7M NH4Ac at a rate of 280 ml / min. The solution then enters the bubble mixer through the "Y"-shaped connector. The bubble mixer is set to a compressed air flow rate of 1.2 L / min. The volume ratio of the pyrolysis solution to solution III is 1:1.

[0129] (5) After neutralization, collect the neutralization reaction solution and centrifuge it at 8000g for 20 minutes to collect the control supernatant, which can be used for the next step of purification.

[0130] The results, obtained by enzyme-linked immunosorbent assay (ELISA), showed that the plasmid concentration in the resuspended bacterial solution was 570 mg / L (calculated from extraction using a plasmid mini-prep kit), and the total plasmid content was 14.36 g.

[0131] After neutralization, 101 L of neutralization reaction solution was obtained. Centrifugation yielded a total of 79.3 L of supernatant. The plasmid concentration in the supernatant was measured to be 116.3 mg / L (HPLC determination), with a lysis yield of 64.2%. Electrophoresis results are as follows... Figure 12 As shown, from Figure 12 It can be seen that the plasmid DNA obtained by the bubble generator in the neutralization process of this comparative example has a similar plasmid concentration to that obtained by the method in Example 1, but has more host RNA. This indicates that the preparation method of this application is superior, easier to scale up, and simpler to operate.

[0132] Comparative Example 2

[0133] Unlike Example 1, the centrifugal pump head impeller used in the second mixing pump in Comparative Example 2 is as follows: Figure 13 As shown, the rest are the same.

[0134] After neutralization, 80 L of neutralization reaction solution was obtained by microplate reader. Centrifugation yielded 66 L of supernatant. The plasmid concentration in the supernatant was measured to be 106.6 mg / L (HPLC determination), and the lysis yield was 64.5%. The lysis yield was lower than that in Example 1.

[0135] Comparative Example 3

[0136] Unlike Example 1, the centrifugal pump head impeller used in the second mixing pump in Comparative Example 3 is as follows: Figure 14 As shown. All other settings are the same.

[0137] Compared with the results of Example 1, the test electrophoresis pattern is as follows: Figure 15 As shown in the figure, the supernatant from the pump head lysis showed a high content of both host DNA and RNA, which is not conducive to plasmid purification.

[0138] Based on the results of the above embodiments, it can be seen that the extraction device of the present invention has sufficient mixing and short mixing time during final product lysis, mild and uniform conditions during neutralization, and after lysis and neutralization, the residual host DNA and RNA are lower than the effect of the bubble mixer, resulting in better product quality. Furthermore, when the speed is moderate, the extracted plasmid DNA has fewer impurities and a high yield.

[0139] This invention innovatively employs a series of mixing components in the alkaline pyrolysis and neutralization stages of plasmid production, combined with a delivery pump, to ensure the pyrolysis and neutralization processes occur in a closed environment, reducing the likelihood of environmental pollution. This facilitates CIP and SIP processes and enables continuous processing, improving production efficiency and simplifying scale-up. Compared to the mainstream Airmix bubble mixer production system, it is easier to scale up, eliminating the need for customized bubble mixers of different sizes, shortening the time required to establish scale-up conditions, and increasing work efficiency. The equipment is simple, easy to operate, and inexpensive, requiring no specialized, customized, or expensive equipment, making it easy to scale up in production and resulting in low production costs. The two mixing units used... This device ensures thorough mixing of bacterial culture and lysis buffer while also providing gentle mixing and neutralization with the neutralizing solution (Solution III), avoiding the need for complex low-shear neutralization equipment. It achieves thorough mixing and shorter mixing time during lysis, and gentle and uniform conditions during neutralization. After lysis and neutralization, the residual host DNA and RNA are lower than those achieved with bubble mixers, resulting in high product quality. Furthermore, the optimized pump chamber size allows for suitable lysis and neutralization time and shear force for product manufacturing, leading to a higher proportion of supercoiled plasmids and less residual host DNA and RNA. Additionally, it eliminates the need for complex multi-stage membrane filtration systems and overnight precipitation after lysis; the equipment can be directly cleaned with CIP, meeting pharmaceutical manufacturing standards while saving process time and reducing costs.

[0140] Furthermore, by optimizing the size of the mixing pump chamber and adjusting the ratio of the pump chamber to the flow rate, the lysis and neutralization time and shear force are made suitable for product production, while also facilitating the scaling up of production. The shape and size of the mixing pump head are optimized, and 3D printing technology is used to design and customize the pump head. While ensuring the mixing effect, the shear force is reduced, preventing host DNA from contaminating the product, and enabling lysis and neutralization to be automated.

[0141] Furthermore, the preparation process does not add high-risk animal-derived ingredients such as RNase, lysozyme, and proteinase K. The production process does not use toxic organic solvents such as isopropanol, phenol, anhydrous ethanol, and other mutagens. The reagents used can be general reagents or meet pharmaceutical grade requirements. No acid neutralization is used, and the requirements for factory equipment are low, making it suitable for large-scale production.

[0142] The above specific embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. An apparatus for extracting plasmid DNA from bacteria, characterized in that, Includes a first mixing component and a second mixing component; The first mixing component and the second mixing component are connected by a pyrolysis spiral tube; at least one liquid inlet is provided on the connecting pipe between the pyrolysis spiral tube and the second mixing component; After the resuspended bacterial solution is mixed in the first mixing component, it is lysed through the lysis spiral tube to obtain a lysate, which is then introduced into the second mixing component to neutralize with solution III to obtain a neutralization reaction solution. The lysate enters the second mixing component through the inlet. The first mixing component and the second mixing component are a first mixing pump and a second mixing pump; The first mixing pump and the second mixing pump include a main shaft, a pump base, a sealing ring, an impeller, and a pump casing. One end of the main shaft is connected to the output end of an external motor, and the other end passes through the center of the pump base through a sealing device and is fixedly connected to the impeller. An annular groove is machined in the contact area between the pump base and the pump casing for installing the sealing ring. Both the first and second mixing pumps have impellers with rear cover plates; the rear cover plates have a plurality of guide columns evenly distributed thereon; the guide columns have an arc-shaped outer surface at least along the direction of impeller rotation. The guide column is perpendicular to the surface of the rear cover plate, and the diameter of the guide column is 2mm-10mm; Solution III includes KAc and NH4Ac, with KAc concentrations ranging from 0.1 mol / L to 6 mol / L and NH4Ac concentrations ranging from 0.2 mol / L to 10 mol / L.

2. The apparatus for extracting plasmid DNA from bacteria according to claim 1, characterized in that, The guide column is one or more of a cylinder or a frustum, or the cross-sectional area of ​​the guide column is the largest in the middle and gradually decreases from the middle to both ends.

3. The apparatus for extracting plasmid DNA from bacteria according to claim 1, characterized in that, The inner diameter of the pyrolysis spiral tube is 0.5cm-15cm; the pump head diameter of the first mixing pump and the second mixing pump is 2cm-100cm; the ratio of the pump chamber volume of the first mixing pump and the second mixing pump to the rated feed volume per minute of a single mixing pump is 1:6-1:

1.

4. The apparatus for extracting plasmid DNA from bacteria according to claim 3, characterized in that, The inner diameter of the pyrolysis spiral tube is 0.5cm-9cm; the pump head diameter of the first mixing pump and the second mixing pump is 4cm-30cm; the ratio of the pump chamber volume of the first mixing pump and the second mixing pump to the rated feed volume per minute of a single mixing pump is 1:6-1:

3.

5. The apparatus for extracting plasmid DNA from bacteria according to claim 1, characterized in that, The inlet and outlet ends of the first and second mixing pumps are both coaxially arranged; the inlet end is located at the center of the pump casing, and the outlet end is located at the center of the pump base.

6. The apparatus for extracting plasmid DNA from bacteria according to any one of claims 1 to 5, characterized in that, The device further includes a filtration assembly, wherein the outlet end of the second mixing assembly is connected to the inlet end of the filtration assembly, and the neutralization reaction solution is filtered through the filtration assembly.

7. The apparatus for extracting plasmid DNA from bacteria according to claim 6, characterized in that, The resuspended bacterial solution includes solution I and bacterial cells containing plasmid DNA. The resuspended bacterial solution is mixed and delivered to the first mixing component by a first delivery pump, and then mixed with solution II delivered to the first mixing component by a second delivery pump before being passed into a lysis spiral tube for lysis. Solution I comprises Tris-HCl and EDTA-2Na, wherein the concentration of Tris-HCl is 2 mmol / L-100 mmol / L, the concentration of EDTA-2Na is 0.1 mmol / L-50 mmol / L, and the pH range of solution I is 6.0-9.0; Solution II comprises NaOH and SDS, wherein the concentration of NaOH is 0.02 mol / L - 5 mol / L and the concentration of SDS is 0.1% - 10%.

8. The apparatus for extracting plasmid DNA from bacteria according to claim 6, characterized in that, The filter assembly structure is one or more combinations of screen type, depth filtration type, and centrifugal filtration type.

9. The apparatus for extracting plasmid DNA from bacteria according to claim 8, characterized in that, The filter assembly has a screen-type or deep-layer filtration structure; the filter pore size is 0.1µm-200µm; and the filter material is cellulose, diatomaceous earth, activated carbon, polypropylene fiber, or silica gel.

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